Protein folding studies are directed towards the goal of achieving the capability of designing novel polypeptide sequences which will fold into desired shapes with medically useful activities. A potentially useful class of synthetic proteins would be small inhibitors of enzymes. Many natural inhibitors consist of globules with a protruding amino acid which resembles the enzyme's substrate, such that the inhibitor competes for enzyme active sites in binding reactions. Enzyme inhibitors regulate many physiological processes such as complement fixation, blood-clotting, ovum fertilization, and cell-cell surface interactions. The objective of the proposed research is to develop strategies for directing formation of specific disulfides in polypeptide chains of de novo design which contain four or more cysteines. These novel rigid internally-crosslinked mainframes could serve as the basis for subsequent inhibitor production by addition of protruding amino acids at different locations on the surface. Experiments will evaluate factors contributing to folding of apamin and endothelin, two proteins with homologous cysteine positions but different sets of disulfides. Disulfide exchange equilibria will be established for synthetic variants having slightly different sequences. Reaction components will be separated by HPLC and characterized with respect to relative yields. Purified components will be analyzed for total mass and for disulfide positions. In a separate project, variants of conotoxin will be designed as possible inhibitors of trypsin and carboxypeptidase. Enzyme inhibition and peptide mapping data will be used to evaluate binding properties and susceptibilities to proteolysis. Molecular modeling calculations will be used to evaluate constraints on loop formation in these projects and in considerations of the sterically restrictive sequences found in immunoglobulin hinge regions. A combination of modeling, disulfide exchange experiments and spectroscopic structural studies will be used to design a disulfide-stabilized alpha-beta motif consisting of several turns of alpha helix covered on one side by a returning extended chain.